Nitric oxide (NO') and eicosanoids are critical mediators of physiological and pathophysiological processes, including inflammation and atherosclerosis. We, and others, have shown that NO'modulates prostaglandin H2 synthase (PGHS, also known as cyclooxygenase) and alters eicosanoid production. We have determined that a particular nitrogen oxide (NOX) species, peroxynitrite (ONOO"), has conflicting effects on PGHS activity: ONOO" can enhance arachidonic acid-stimulated PGHS activity, but can also deactivate the enzyme via tyrosine (Tyr) nitration. In addition, S-nitrosation of cysteine (Cys) residues by certain NOX species activate PGHS by unknown mechanisms. The hypothesis central to the studies proposed is that NOX species alter PGHS-1, PGHS-2 and prostacyclin synthase (PGI2S) activities and that alteration of eicosanoid synthesis will impact both atherosclerotic lesion development and thrombosis. In Specific Aim 1, we propose to determine how S-nitrosation of Cys residues impacts on Tyr radical formation. We also propose to determine mechanisms of Tyr nitration in PGHS-1 and PGHS-2 following exposure to NOX species under conditions of oxidative stress. We predict that heme directs nitration of internal Tyr residues critical to catalytic activity that will abolish enzyme function, whereas nitration of surface-exposed Tyr residues (by a heme-independent mechanism) will have little impact. In collaboration with Dr. S. Gross (Project 4) we will determine specific Tyr sites nitrated by different NOX species in purified enzyme, cells and in lesions from a mouse model of atherosclerosis. We will be able to draw conclusions concerning the mechanism of nitration in atherosclerosis, which will enable us to design key studies to reduce inflammatory oxidative damage to vascular enzymes. In Specific Aim 2, we propose to determine the role of iNOS in PGHS-dependent eicosanoid production during atherosclerosis. We propose that in the absence of iNOS, PGHS may assume a compensatory role and provide an increase in beneficial eicosanoids that lead to a reduction in atherosclerosis observed in ApoE^iNOS"'" mice compared to ApoE"'" mice. In collaboration with Dr. A. Marcus (Project 1), we propose to measure eicosanoid levels in aortic tissue obtained from ApoE"'" and ApoE'^iNOS"'" mice. Using COX inhibitors, we will identify the specific roles of PGHS-1 and PGHS-2-derived eicosanoids in the development of atherosclerosis in ApoE"A and ApoE^'iNOS"'" mice. Finally, in collaboration with Dr. K. Hajjar (Project 2), we will define the role of PGHS and PGI2S-derived eicosanoids in the development of arterial thrombosis in mice lacking iNOS. Results from these studies will define the modulatory effects of NOX species on PGHS function and the regulation of eicosanoid biosyntheses as well as the impact of these mediators on processes related to atherosclerosis and thrombosis.